Single-stranded DNA (ssDNA) viruses cause severe disease problems in plants and animals (Moffat (1999) Science 286:1835). Geminiviruses and nanoviruses infect many important crops worldwide, such as cassaya, bean, pepper, tomato, sugar beet, cotton and maize (Brown and Bird (1992) Plant Disease 7:220-225; Czosnek and Laterrot (1997) Arch. Virol. 142:1391-1406; Lotrakul, et al. (1998) Plant Dis. 82:1253-1257; Zhou, et al. (1997) J. Gen. Virol. 78:2101-2111; Mansoor, et al. (1999) Virology 259:190-199; Polston, et al. (1999) Plant Dis. 83:984-988). Circoviruses cause significant disease losses among livestock and poultry (Allan, et al. (1998) J. Vet. Diagn. Invest. 10:3-10; Bassami, et al. (1998) Virology 249:453-9; Nayar, et al. (1999) Can. Vet J. 40:277-8). A human circovirus in Hepatitis C patients has also been identified (Miyata, et al. (1999) J. Virol. 73:3582-3586 Mushahwar, et al. (1999) Proc. Natl. Acad. Sci. USA. 96:3177-3182). Even though these viruses have diverse host ranges and cause different diseases, they are highly related to each other.
Geminiviruses, nanoviruses, and circoviruses amplify their circular ssDNA genomes via a rolling circle mechanism through the combined action of a single viral protein, Rep, and the host DNA replication machinery (Laufs, et al. (1995) Biochimie 77:765-773; Mankertz, et al. (1997) J. Virol. 71:2562-2566; Katul, et al. (1998) J. Gen. Virol. 79:3101-3109; Mankertz, et al. (1998) J. Gen. Virol. 79:381-384; Hanley-Bowdoin, et al. (1999) Crit. Rev. Plant Sci. 18:71-106). Rep initiates plus-strand DNA synthesis by cleaving the viral origin within a hairpin structure at an invariant sequence, acts as a DNA ligase to terminate rolling circle replication, and hydrolyzes ATP. Because of the functional conservation, Rep proteins from all three ssDNA virus families are highly homologous.
The Geminiviridae family is classified into four genera based on genome structure, insect vector and type of host (Rybicki 1994; Briddon, Bedford et al. 1996). The four genera infect a broad range of plants and cause significant crop losses worldwide (Brown and Bird 1992; Brown 1994; Rybicki and Pietersen 1999; Morales and Anderson 2001; Mansoor, Briddon et al. 2003). All geminiviruses are characterized by twin icosahedral capsids (Zhang, Olson et al. 2001; Bottcher, Unseld et al. 2004) and single-stranded DNA (ssDNA) genomes that replicate through double-stranded DNA (dsDNA) intermediates (Hanley-Bowdoin, Settlage et al. 1999).
Geminiviruses replicate their small, circular DNA genomes using a combination of rolling circle and recombination-mediated replication (Gutierrez 1999; Jeske, Lutgemeier et al. 2001). They encode the proteins required for initiation of replication, Geminivirus Replication Initiation Protein (Rep), and depend on host polymerases for DNA synthesis (Gutierrez 2000; Hanley-Bowdoin, Settlage et al. 2004). Much of our knowledge of geminivirus replication comes from studies of TGMV, a typical begomovirus with a bipartite genome. Two of the seven proteins encoded by TGMV are involved in viral replication. AL1 is required for viral replication (Elmer, Brand et al. 1988; Hanley-Bowdoin, Elmer et al. 1990), whereas AL3 is an accessory factor that enhances viral DNA accumulation (Sunter, Hartitz et al. 1990). The AL1 protein shows conservation across all four genera. Different nomenclatures have been used to designate AL1, which is also known as Rep, AC1 or C1. As used herein, the Rep designation is employed because it is applicable to all geminiviruses.
Rep is a multifunctional protein that mediates both virus-specific recognition of its cognate origin (Fontes, Eagle et al. 1994) and transcriptional repression (Eagle, Orozco et al. 1994; Eagle and Hanley-Bowdoin 1997). Rep initiates and terminates (+) strand DNA synthesis within a conserved hairpin motif (Heyraud-Nitschke, Schumacher et al. 1995; Laufs, Traut et al. 1995; Orozco and Hanley-Bowdoin 1996). It also induces the accumulation of host replication factors in infected cells (Nagar, Pedersen et al. 1995). Rep binds to dsDNA at a repeated sequence in the origin (Fontes, Eagle et al. 1994; Fontes, Gladfelter et al. 1994), cleaves and ligates DNA within an invariant sequence of a hairpin loop (Laufs, Jupin et al. 1995; Orozco and Hanley-Bowdoin 1996), and is thought to unwind viral DNA in an ATP-dependent manner (Gorbalenya and Koonin 1993; Pant, Gupta et al. 2001). Rep interacts with itself and AL3 (Settlage, Miller et al. 1996). It binds to several host factors involved in DNA transactions, including the replicative clamp PCNA (Castillo, Collinet et al. 2003), the clamp loader RFC (Luque, Sanz-Burgos et al. 2002), histone H3 and a mitotic kinesin (Kong and Hanley-Bowdoin 2002). Rep also interacts with host regulatory factors, including the retinoblastoma protein (pRBR) which modulates the a cell cycle and differentiation (Xie, Suareziopez et al. 1995; Grafi, Burnett et al. 1996; Ach, Durfee et al. 1997), a novel protein kinase (GRIK) associated with leaf development (Kong and Hanley-Bowdoin 2002), and Ubc9—a component of the sumoylation pathway (Castillo, Kong et al. 2004).
The functional domains of Rep have been mapped by deletion and mutational studies (FIG. 1). The N-terminal half of Rep contains overlapping domains for DNA cleavage/ligation, DNA binding, and protein interactions (Orozco, Miller et al. 1997; Orozco and Hanley-Bowdoin 1998). NMR spectroscopy revealed that the overlapping DNA binding/cleavage domains contain a β-sheet cluster that resemble other nucleic acid binding proteins (Campos-Olivas, Louis et al. 2002). The characterized Rep protein interactions fall into two classes—proteins that bind between amino acids 101-180 (Kong, Orozco et al. 2000; Settlage, Miller et al. 2001) and those that bind between amino acids 134-180. (Orozco, Kong et al. 2000; Kong and Hanley-Bowdoin 2002). The putative DNA helicase domain is in the C-terminus (Gorbalenya and Koonin 1993; Pant, Gupta et al. 2001).
Rep contains several conserved amino acid and structural motifs (FIG. 1). Motifs I, II and III are characteristic of rolling circle initiators (Ilyina and Koonin 1992; Koonin and Ilyina 1992). Motif I (FLTY) is a determinant of dsDNA binding specificity (Chatterji, Chatterji et al. 2000; Arguello-Astorga and Ruiz-Medrano 2001). Motif II (HLH) is a metal binding site that may impact protein conformation and/or catalysis. Motif III (YxxKD/E) is the catalytic site for DNA cleavage with the hydroxyl group of the Y residue forming a covalent bond with the 5′ end of the cleaved DNA strand (Laufs, Traut et al. 1995). The aromatic ring of the Y residue plays a role in dsDNA binding (Orozco and Hanley-Bowdoin 1998). The three motifs are exposed and in close proximity on the β-sheet surface in the Rep N-terminus (Campos-Olivas, Louis et al. 2002). Other conserved motifs include a sequence of near identity and unknown function immediately C-terminal of Motif III (Kong, Orozco et al. 2000), a helix-loop-helix motif that mediates pRBR binding (Arguello-Astorga, Lopez-Ochoa et al. 2004), and a NTP binding consensus (Walker, Saraste et al. 1982).
A variety of strategies have been applied to geminivirus resistance, including conventional breeding and transgenic approaches. Conventional breeding has been confounded by the limited sources of natural resistance, the multigenic nature of the resistance traits, and the time required for a breeding program (Miklas, Johnson et al. 1996; Pessoni, Zimmermann et al. 1997; Velez, Bassett et al. 1998; Weiz, Schechert et al. 1998; Kyetere, Ming et al. 1999). TYLCV resistance genes have been introgressed from a wild Lycopersicon species (Pilowsky and Cohen 1990; Lapidot, Friedmann et al. 1997; Friedmann, Lapidot et al. 1998; Vidavsky and Czosnk 1998). This resistance is often unsatisfactory due to linkage with poor fruit quality, complex inheritance patterns, and the difficulty of transfer to commercial cultivars. Most conventional resistances collapse under early or severe infection pressure (Lapidot and Friedmann 2002). There is also evidence that host resistance genes are not equally effective against different geminiviruses (Pernet, Hoisington et al. 1999; Pernet, Hoisington et al. 1999), and many host genes only confer tolerance (Gilreath, Shuler et al. 2001; Lapidot, Friedmann et al. 2001; Gomez, Pinon et al. 2004). Tolerant plants, which support viral replication—albeit at lower levels, can serve as reservoirs for mutant and recombinant viruses that have the potential to overcome resistance.
Several transgenic strategies based on pathogen-derived resistance have also been tested. There is one report of transgenic tomatoes that contain a mutant begomovirus coat protein gene and display tolerance (Kunik, Salomon et al. 1994), but this result has not been reproduced by other researchers using wild type viral sequences (Azzam, Diaz et al. 1996; Sinisterra, Polston et al. 1999). Instead, expression of geminivirus sequences frequently results in the production of functional proteins that typically complement defective viruses or cause symptoms (Hanley-Bowdoin, Elmer et al. 1989; Hayes and Buck 1989; Hanley-Bowdoin, Elmer et al. 1990; Pascal, Goodlove et al. 1993; Latham, Saunders et al. 1997; Krake, Rezaian et al. 1998; Guevara-Gonzalez, Ramos et al. 1999; Hou, Sanders et al. 2000:Sunter, 2001 #7731). The reduced sensitivity to pathogen-derived resistance may reflect the lack of an RNA genomic form and the ability of geminiviruses to modulate host gene silencing (Ratcliff, Harrison et al. 1997; Voinnet, Pinto et al. 1999; Covey and AlKaff 2000; Noris, Lucioli et al. 2004; Vanitharani, Chellappan et al. 2004). Antisense RNA and defective-interfering replicon strategies have also been of limited success (Stanley, Fischmuth et al. 1990; Day, Bejarano et al. 1991; Frischmuth and Stanley 1994; Aragao, Ribeiro et al. 1998; Asad, Haris et al. 2003). Recent reports suggested that RNAi constructs can confer strong resistance, but this strategy is limited to homologous (or very closely related) geminiviruses (Pooggin, Shivaprasad et al. 2003; Pooggin and Hohn 2004). Transgenic plants that inducibly express dianthin upon geminivirus infection also display resistance (Hong, Saunders et al. 1996), but the safety of a toxic ribosome-inactivating protein has not been established. Expression of mutant begomovirus movement proteins in transgenic plants also resulted in resistance, but the phenotype is variable possibly because of the ability of the mutant proteins to confer symptoms in the absence of infection (Pascal, Goodlove et al. 1993; Duan, Powell et al. 1997; Duan, Powell et al. 1997; Hou, Sanders et al. 2000).
Unlike the strategies described above, Rep mutants have proven effective at interfering with geminivirus replication in cultured cells. Mutations in Motif III, the ATP binding site and the oligomerization domain (FIG. 1) interfere with virus replication in transient assays (Hanson and Maxwell 1999; Orozco, Kong et al. 2000; Chatterji, Beachy et al. 2001). However, plants that stably produce the Rep protein display developmental defects (Brunetti, Tavazza et al. 1997; Brunetti, Tavazza et al. 2001), and expression is selected against during meiosis. The PRBR protein is required for gametogenesis (Ebel, Mariconti et al. 2004), suggesting that the Rep expression problem reflects its interaction with pRBR. Recent experiments showed that inclusion of a pRBR binding mutation in an interfering Rep transgene results in stable expression through at least 3 generations. Because Rep is highly specific for its cognate viral origin (Fontes, Gladfelter et al. 1994; Chatterji, Chafterji et al. 2000), the same plants were designed to also express a mutant AL3 in an effort to the broaden resistance. (AL3 functions in virus-nonspecific manner to enhance viral accumulation (Sunter, Stenger et al. 1994; Sung and Coutts 1995)). The plants coexpressing the mutant Rep and AL3 proteins are immune to infection by the homologous virus through at least three generations. It is not yet known if they are resistant to unrelated begomoviruses. In contrast, other studies showed that infection with a homologous virus can lead to Rep transgene silencing. It is desirable to develop alternative resistance strategies.
Peptide aptamers resemble single chain antibodies, but because of their in vivo selection, are more likely to be stably expressed and correctly folded and to interact with their targets in an intracellular context (Crawford, Woodman et al. 2003). If an aptamer binds to residues critical for function, it can inactivate its target and interfere with cellular processes. For example, an aptamer that binds to the active site of the cell cycle regulator, cdk2, was isolated by screening a combinatorial peptide library in yeast dihybrid assays (Colas, Cohen et al. 1996). The aptamer blocks cdk2/cyclin E kinase activity in vitro and, when expressed in vivo, retards cell division (Cohen, Colas et al. 1998). An aptamer that interacts with the dimerization domain of cell cycle-associated transcription factor, E2F, also interferes with cell cycle progression in animal cells (Fabbrizio, LeCam et al. 1999). Aptamers have also been expressed in flies to study the specific roles of cdk1 and cdk2 during Drosophila organogenesis (Kolonin and Finley 1998). They have been used to distinguish between and selectively inactivate allelic variants of Ras and to inhibit Rho GTP exchange factors (Schmidt, Diriong et al. 2002; Xu and Luo 2002; Kurtz, Esposito et al. 2003) as well as interfere with the EGF signaling pathway by binding to the downstream transcription factor—Stat3 (Buerger, Nagel-Wolfrum et al. 2003; Nagel-Wolfrum, Buerger et al. 2004).
Peptide aptamers are especially well suited for targeting noncellular factors like viral proteins. An aptamer that binds to the hepatitis B virus core protein and inhibits viral capsid formation and replication has strong antiviral activity in liver cells (Butz, Denk et al. 2001). Aptamers that target the E6 or E7 proteins of human papillomavirus and block their anti-apoptotic activities result in specific elimination of HPV-positive cancer cells (Butz, Denk et al. 2000; Nauenburg, Zwerschke et al. 2001).
The present inventors have found that expression of aptamers that target essential, conserved Rep motifs can interfere with viral replication and confer broad resistance against geminivirus infection.